Binding specificity of hit compounds as determined by direct-binding analysis using SPR. FIXa (black bars), FXa (dark gray bars), prothrombin (striped), or FV (white) were tested for their ability to bind to a 10/90 PS/PC phospholipid membrane in the presence of fixed concentrations of 10 µM of hit molecules. For comparison also, the binding of FVIII (in light gray) at this concentration was included. Residual binding was calculated by division of the binding signal in the presence of compound over the signal in the absence of compound (DMSO only), while correcting for binding to 100% PC membranes. Positive controls were included in the assay to ascertain the correctness of the assay outcome. As a positive control runs for FIXa, FXa and prothrombin were performed in the presence of 20 mM of EDTA, whereas for FV and FVIII, an inhibitory antibody directed against the C2 domain was used at a concentration 0.25 mg/mL. Data shown are averages of at least 2 individual measurements, with the standard error of the mean being <15% for all values.